Project Summary-Abstract Organisms living on Earth have to cope with daily changes in their environment. The circadian clock is an endogenous oscillator, with a period of approximately 24 hours, which enables organisms to coordinate metabolism, physiology, and development in anticipation of diurnal and seasonal environmental changes therefore enhancing their fitness. The intricate network underlying the circadian oscillator at the molecular level has long been thought to rely on interlocked transcriptional-translational feedback loops. However, additional layers of regulation at the cellular and organismal level are required to explain the complex behavior of the system. To unravel this wiring and elucidate the role of key clock components, in this proposal we plan to combine a set of biochemical, genetic, functional genomic, and bioinformatic approaches. TOC1 is an essential player in the core oscillator and recent work in our laboratory suggests that it can bind to RNA. By solving the atomic structure of TOC1 we seek to dissect its molecular properties. This will be instrumental to comprehend its regulatory mechanism and its function maintaining plant rhythmicity and regulating the expression of output genes. GI is also an oscillator component required for the proper ticking of the circadian clock and acts as a key regulatory hub directly connecting the clock to a plethora of physiological processes. To investigate the pivotal role of GI in the regulation of output pathways we propose to uncover and characterize its interactome network through molecular, genetic and proteomic analyses. These results will shed light on its regulatory function in key physiological processes providing important advances in our understanding of how the circadian clock impacts plant development. Finally, it has been recently revealed that the plant clock might have a central oscillator, similar to the mammalian system, able to coordinate peripheral clocks. To examine the organization of the plant circadian system, we will perform a genome-wide tissue- specific expression analysis with exceptional spatio-temporal resolution, complementing it with a phenotypical characterization. Altogether, the approach outlined here will expand our understanding of tissue and organ specific clocks and their architecture to regulate physiological and developmental processes. The knowledge gained from this proposal will contribute to develop a comprehensive view of the circadian clock as a network expanding from the molecule to the organismal level. This concept can be combined with insights from other organisms to improve our interpretation of circadian biology, ultimately impacting research in human health and circadian associated disorders.